The long-term goal of this project is to understand the cellular and molecular mechanisms that control spermatogonial differentiation in the rhesus monkey, a representative higher primate. The strategy underlying the proposal is based, in part, on the finding that while spermatogonial proliferation occurs in the absence of gonadotropin stimulation, differentiation of primate spermatogonia occurs only in the presence of a gonadotropin drive. This difference in the endocrine requirements of the two processes will be used to interrogate the testicular genes, gene networks and any novel transcripts that determine the decision of spermatogonia to renew or to differentiate. There are three major questions. First, what genes and pathways within spermatogonia underlie the decision of the cell to differentiate rather than undergo self-renewal. Second, what are the gonadotropin dependent Sertoli cell paracrine signals that instruct undifferentiated spermatogonia to proceed down the path of differentiation. Third, to what extent is spermatogonial differentiation dependent on Vitamin A. To begin to answer these questions, RNA-seq will be used to assess global changes in germ cell and Sertoli cell gene expression and transcriptome profiles associated with the initial step in placing undifferentiated type A spermatogonia on the gonadotropin dependent pathway of differentiation. Following confirmation by qRTPCR, cell specific expression of those genes and relevant transcripts that are shown by qRTPCR to be up- or down-regulated in association with spermatogonial differentiation will then be determined using immunohistochemistry (IHC) and/or in situ hybridization (ISH). The effects of Vitamin A deficiency (VAD) on spermatogonial proliferation will be determined by feeding the animals a Vitamin A deficient diet. Two Specific Aims will be addressed; 1 to identify germ cell and Sertoli cell genes, gene networks and novel transcripts associated with the decision of undifferentiated type Ap spermatogonia to commit to a pathway of differentiation, and 2 To determine whether the decision of undifferentiated Type Ap spermatogonia to commit to the path of differentiation in the monkey is dependent on Vitamin A. This work will be conducted in an interactive intellectual environment that is generated by a group of investigators interested in related aspects of spermatogonial biology and Sertoli cell function. It is anticipated that the proposed work will provide fundamental insight into spermatogonial differentiation: a critical aspect of spermatogenesis that will be relevant to the treatment of infertility in the human male and to the development of novel male contraceptives. PUBLIC HEALTH RELEVANCE: The understanding of the molecular bases underlying the decision of undifferentiated spermatogonia to proceed down the pathway of differentiation or to renew themselves is important because this is a key step in the process of spermatogenesis. To date, this step in the spermatogenic lineage has been investigated primarily to rodents, and studies of non-human primates are urgently needed.